Biomedical engineers at North Carolina’s Duke University have used 3D printing to create a diagnostic tool that can detect signs of disease from a single drop of blood. The “D4 assay” consists of an inket-printed array of antibodies and a 3D printed smartphone attachment.

Although there are many ways to test for diseases, the presence of certain antigens in our blood can be one of the clearest signs that something is amiss with the body. That’s why biomedical engineers at Duke have made an easy-to-use “lab on a chip” that can be used to detect low levels of antigens, even in settings that lack standard laboratory testing equipment.

In a study published in the Proceedings of the National Academy of Sciences, the researchers explain how the D4 assay allows clinicians to avoid the slowness of other devices without sacrificing sensitivity or accuracy. The device can identify a disease biomarker in just 15 minutes, though longer incubation times can be used if further sensitivity is needed.

Importantly, the information collected by the device can be easily read using a tabletop scanner or a 3D printed smartphone attachment that improves a phone’s onboard camera. This smartphone feature is especially useful for deploying the D4 assay in point-of-care settings.

"The D4 assay enables us to conduct high-performance diagnostic testing with minimal resources, making it a promising platform for increasing access to sensitive and quantitative diagnostic tools," said Angus Hucknall, co-author of the paper.

The D4 assay follows the lead of existing tools in using a matched pair of antibodies to detect and capture a target protein in a blood sample. The array contains both immobilized “capture” antibodies and soluble “detection” antibodies, which are tagged with a fluorescent marker to allow the researchers to identify how much of the antigen is present.

Adding a drop of blood to the slide causes the detection antibodies to dissolve, separate from the array, and bind to the targeted proteins in the blood. These antibody-protein pairs, illuminated by the fluorescent marker, then attach to the capture antibodies that remain on the slide.

Existing diagnostic tests like the enzyme-linked immunosorbent assay (ELISA), which is used to detect diseases like Zika and HIV, have proven themselves effective in many ways, and the D4 assay is similar to the ELISA in that it uses a pair of matched antibodies.

Unlike the ELISA, however, the D4 assay's antibody array is printed on a novel polymer brush coating which works like Teflon to prevent unwanted proteins from attaching to the slide. By stopping unwanted proteins from sticking to the assay, it becomes easier to detect the targeted ones.

“The real significance of the assay is the polymer brush coating," said Ashutosh Chilkoti, chair of the Department of Biomedical Engineering (BME) at Duke and senior author on the paper. “The polymer brush allowed us to store all of the tools we need on the chip while maintaining a simple design.”

The Duke biomedical engineers conducted a clinical trial to assess the performance of the D4 assay, measuring the serum leptin levels in patients at Duke University Medical Center and comparing them to those observed with the clinical ELISA platform. They found that the results from the D4 assay were on par with those from the ELISA test.

But the D4 assay purportedly offers a number of advantages over assays like the ELISA. For one, the slide simply needs to be washed in a buffer to remove extraneous particles—there’s no need for a more complicated process. It’s also highly portable, because the dry slides don't need to be refrigerated during transport.

The researchers involved in the study estimate that D4 chips will cost less than one dollar each, with the 3D printed mobile phone attachment less than $30 when produced in bulk.

The next stage of the project will involve taking the D4 assay to Liberia to test its efficiency in the field. The team will try to figure out how the D4 assay can be used to monitor and plan treatment strategies for malnutrition.